Am. J. Trop. Med. Hyg., 00(0), 2020, pp. 1–4 doi:10.4269/ajtmh.19-0180
Copyright © 2020 by The American Society of Tropical Medicine and Hygiene
Decline in Total Serum IgE and Soluble CD30 in the Context of Soil-Transmitted Helminth Decline
in Bolivia
Chiara Della Bella,1† Michele Spinicci,1† David Rojo,2Alessia Grassi,1Herlan Gamboa,3Marisa Benagiano,1Roberto Torrez,4 Simona Tapinassi,1Simona Gabrielli,5Gabriella Cancrini,5Fabio Macchioni,6Heba Alnwaisri,1Annalisa Azzurri,1
Joaqu´ın Monasterio,4Antonio Montresor,7Piero Olliaro,8Mario Milco D’Elios,1*‡ and Alessandro Bartoloni1*‡
1
Dipartimento di Medicina Sperimentale e Clinica, Universit `a degli Studi di Firenze, Firenze, Italy;2Escuela de Salud del Chaco Tekove Katu, Gutierrez, Plurinational State of Bolivia;3Facultad Integral del Chaco, Universidad Aut ´onoma Gabriel Ren ´e Moreno, Camiri, Plurinational State of Bolivia;4Servicio Departamental de Salud (SEDES) de Santa Cruz, Santa Cruz, Plurinational State of Bolivia;5Dipartimento di Sanit `a Pubblica e
Malattie Infettive, Universit `a di Roma Sapienza, Roma, Italy;6Dipartimento di Scienze Veterinarie, Universit `a degli Studi di Pisa, Pisa, Italy; 7
Department of Control of Neglected Tropical Diseases (NTD), World Health Organization, Geneva, Switzerland;8Nuffield Department of Medicine, Centre for Tropical Medicine and Global Health, University of Oxford, Oxford, United Kingdom
Abstract. In the Bolivian Chaco, recent surveys documented a dramatic decrease in the prevalence of soil-transmitted helminth (STH) infections as compared with the 1980s after thirty years of preventive chemotherapy (PC). Concomitant immunological rearrangements are expected. Because nematode infections are associated with increased levels of circulating IgE and glycoprotein CD30 soluble form (sCD30), this study aims to evaluate changes in serological markers of T helper (Th)2-cells activity between 1987 (high STH prevalence) and 2013 (low STH prevalence) in rural communities in the Bolivian Chaco area. We collected 151 sera during two different surveys in 1987 (n = 65) and 2013 (n = 86) and
measured the concentration of total IgE and sCD30 by immunoassays. We found a statistically significant
age-independent decrease in the total IgE (P < 0.0001) and sCD30 (P < 0.0001) from 1987 to 2013. The significant decrease in serological Th2 markers (IgE and sCD30) between 1987 and 2013 is consistent with the drop in STH prevalence in this geographical area during the same period of time. Further studies might elucidate the clinical and epidemiological impact of these serological rearrangements
INTRODUCTION
It is estimated that soil-transmitted helminthes (STHs), namely, the roundworm Ascaris lumbricoides, the whipworm Trichuris trichiura, and the hookworms Necator americanus and Ancylostoma duodenale, infect approximately one-fourth to one-fifth of the world’s population. Children are particularly exposed: approximately 270 million preschool-age children (pre-SAC) and more than 550 million school-age children (SAC) are affected, mostly living in resource-deprived areas, where these parasites are extensively transmitted because of
the low socio-sanitary conditions.1 Moreover, 370 million
persons are now estimated to be infected worldwide with another intestinal nematode, Strongyloides stercoralis, but the global prevalence of this parasite has long been under-estimated, as it has been based on studies using poorly sen-sitive diagnostics.2
In 2001, the WHO endorsed a resolution to control STH infections through improved sanitation, health education, and preventive chemotherapy (PC), whereby anthelmintic drugs such as albendazole or mebendazole should be periodically administered to high-risk groups, such as preSAC, SAC, and women of reproductive age.3The global target is to eliminate morbidity due to soil-transmitted helminthiases in children by 2020, by regularly treating at least 75% of the children in en-demic areas1; in 2017, the coverage has reached more than
69% of the SAC and preSAC groups.4
In Bolivia, PC based on 6-monthly single-dose mebenda-zole delivery to SAC has been implemented since 1985 and
achieved impressive results, at least in the Chaco region, in the southeastern part of the country. Parasitological surveys conducted 26 years apart in rural areas of the Bolivian Chaco documented a dramatic decrease in STH prevalence between 1987 and 2013: the prevalence of hookworm dropped from up
to 50% to 0.4–1.3%, A. lumbricoides from 19% to 0.9–1.5%,
and T. trichiura from 19% to 0%.5–7Based on thesefindings, and according to WHO recommendations, in September 2016, local authorities interrupted PC delivery in this area and started a program of STH surveillance through annual cross-sectional parasitological surveys at sentinel sites. Two years
later, surveillance confirmed the sustained STH reduction
(< 1%) in the entire ecological zone.8
Concomitantly, a serosurvey evidenced a drop of anti–
S. stercoralis antibody prevalence from 1987 to 2013 (from 16% to 6%), although PC is not effective against this para-site.9Conversely, protozoan parasitic infections did not show any substantial decreasing.7
Parasitic infections are associated with type 2 immune re-sponses, characterized by T helper (Th)-2 cells activity, eo-sinophilia, and elevated IgE levels.10The release by Th-2 cells of high amounts of soluble CD30 (sCD30), belonging to the tumor necrosis factor/nerve growth factor receptor super-family, is also associated with type 2 response.11Type 2 im-mune response is also involved in allergic diseases, which can lead to debilitating or even fatal disorders. Studies in animals provide evidence that helminth infections downregulate parasitic-specific immune responses and protect against al-lergic airway inflammation.12According to the so-called hy-giene hypothesis, the increase in allergic disorders observed in western countries in recent times would be related to the low exposure in childhood to microbes and parasites, which would
modulate the human immune system growth.13
Taking advantage of the preferential expression of IgE and sCD30 as Th2 activation markers,14we investigated whether a
* Address correspondence to Mario Milco D’Elios or Alessandro Bartoloni, Dipartimento di Medicina Sperimentale e Clinica, Universit `a degli Studi di Firenze, Largo Brambilla, 3, Firenze 50134, Italy. E-mails: mariomilco.delios@unifi.it or alessandro.bartoloni@unifi.it
† These authors contributed equally to this work. ‡ These authors contributed equally to this work.
change in serum concentration of IgE and sCD30, as a proxy for Th2 activation markers, had occurred over a 26-year period (from 1987 to 2013), among people living in two rural com-munities of the Bolivian Chaco area at the same time of the progressive disappearance of STH infections.
MATERIALS AND METHODS
Study area and population. The Bolivian Chaco is a semi-arid and sparsely populated region, located in the southeast of Bolivia (longitude 64°300 and 58°500 west of the Greenwich meridian; latitude 17°580 and 22°200 south). Serum samples were collected within research activities conducted in 1987 in Javillo, a very small community (of about 110 inhabitants), and in 2013, in the rural community of Ivamirapinta (about 1,200 in-habitants). Both communities lie in the municipality of Gutierrez (Cordillera Province, Department of Santa Cruz).
Determination of total IgE and sCD30 in human sera. Sera were obtained from venous blood samples, stored
at −20°C in Bolivia, shipped within 2–4 weeks to Florence,
Italy, and stored here at−70°C until tested. Sera were ano-nymized and labeled with a unique code, allowing to retrieve demographic information.
The total IgE level in the human serum samples was de-termined via ImmunoCAP total IgE kit automatically
pro-cessed into the Phadia 250 instrument (Thermo Scientific,
Massachusetts). Inside the instrument, the anti-IgE antibody, covalently linked to the solid phase, reacts with the IgE in the serum samples. After incubation and a washing step, enzyme-labeled anti-IgE antibody is added to form a complex. A further washing step, necessary for the removal of unbound sub-stances, is carried out before a second incubation period with the developing agent. Finally, the reaction is stopped and the eluate’s fluorescence measured. The total IgE amount in each tested sample is then available in kU/L.
Sera-soluble CD30 concentrations were determined using Invitrogen ZyQuick sCD30 ELISA kit (Invitrogen Corporation, Camarillo, California), according to the manufacturer’s in-structions. Briefly, soluble CD30 in the samples and standards binds to the anti-sCD30 coated on the plate. A biotin-conjugated antibody is added to bind the sCD30 captured by thefirst antibody. After adding Streptavidin-HRP and the sub-strate solution, the colorimetric reaction is terminated by the addition of acid. Absorbance was measured at 450 nm with Multiskan GO spectrophotometer and analyzed with its SkanIT 3.2 software (Thermo Fisher Scientific, Massachusetts).
Statistical analysis. Statistical analysis was performed with STATA 11.0 (StataCorp, College Statio, TX). Two age categories were created: < 26 and³ 26 years of age (the time difference between the two surveys, so as to have a group of
2013 subjects who were not yet born when thefirst survey was conducted). Descriptive statistics was used for the calculation of means, SD, medians, and interquartile range of quantitative data. The normal distributions of test results for the two groups of sera were compared using the Student’s t-test. Differences in age distribution were estimated by the Mann–Whitney test. Linear regression models were performed to establish the association between serum concentration of Th2 markers and year of collection, allowing for age (continuous). P < 0.05 was considered statistically significant.
Ethics statement. The studies were programmed and conducted in agreement with the Ministry of Health of the Plurinational State of Bolivia (within the Convenio Ministerio de Salud y Deportes, Estado Plurinacional de Bolivia/C ´atedra de Enfermedades Infecciosas, Universidad de Florencia, Ita-lia) and with the support of the Guaran´ı political organization (Asamblea del Pueblo Guaran´ı). The study was approved by a local Ethics Committee (Colegio M ´edico de Santa Cruz, TDEM CITE No. 028/2017).
RESULTS
Overall, 151 sera were collected from people living in two rural communities (age 4–70 years): Javillo (65 serum samples, collected in 1987) and Ivamirapinta (86 serum samples, col-lected in 2013). Table 1 summarizes the characteristics of the participants in the surveys. No significant difference in sex distribution was observed between 1987 and 2013 population; participants were significantly older in 2013 (P < 0.0001).
The mean serum total IgE concentrations were 205.4 kU/L (SD 55.8 kU/L, median 186.0 kU/L) and 77.9 kU/L (SD 21.3 kU/ L, median 81.5 kU/L) in 2013 and 1987, respectively. The mean serum total sCD30 concentrations were 46.0 U/mL (SD 19.2 U/mL, median 41.0 U/mL) and 18.4 U/mL (SD 10.6 U/mL, median 15.0 U/mL) in 1987 and 2013, respectively. The dif-ference was statistically significant for both total IgE and sCD30 (P < 0.0001) (Figure 1), both overall and in each of the age categories (SAC, and subjects aged < 26 and³ 26 years) (Table 2). By linear models, both IgE and sCD30 confirmed a significant decline from 1987 to 2013 (P < 0.0001), indepen-dent of age (Supplemental Table). No significant differences were found in serum concentration of either markers be-tween SAC and bebe-tween subjects aged < 26 and³ 26 years, both in 1987 and in 2013 (Table 2).
DISCUSSION
Our study shows a significant decrease in IgE and sCD30
concentrations (P < 0.0001) between sera collected 26 years apart in two rural communities of the Bolivian Chaco. These
TABLE1
Demographic data of the 151 subjects surveyed in 1987 and 2013 in the Bolivian Chaco
Javillo (1987)
Ivamirapinta (2013)
Gender Total N = 65 N = 86
Male 48% (<I>N</I> = 31) 48% (<I>N</I> = 41) Female 52% (<I>N</I> = 34) 52% (<I>N</I> = 45) Age (years) Median (IQR) range 15.5 (10–30) 4–70 32 (13–48) 6–70
< 26 71% (<I>N</I> = 46) 43% (<I>N</I> = 37) ³ 26 29% (<I>N</I> = 19) 57% (<I>N</I> = 49) SAC 51% (<I>N</I> = 33) 29% (<I>N</I> = 25)
IQR = interquartile range; SAC = school-age children (4–15 years old).
changes paralleled an impressive drop in STH prevalence in this area, from more than 50% to < 1%, likely the result of 30 years of PC.8Of note, no STH infections were identified in Ivamirapinta in 2013 during a parasitological survey, which involved 111 inhabitants (median age 30 years, range 1–73).15 During the same period, a significant decrease in seropreva-lence was documented also for S. stercoralis, although no large-scale control measures potentially affecting strongyloi-diasis were implemented.9No significant change in intestinal
protozoan parasite infections was observed during the same time interval, indicating socioeconomic conditions have not substantially improved during that period. In the two com-munities surveyed, the seropositivity for S. stercoralis ob-served in Javillo in 1987 was 12%, and 7% in Ivamirapinta in 2013 (unpublished data). No recent data are available on the prevalence of other helminth infections potentially affecting Th type 2 response, in the Bolivian Chaco. A high rate of
Man-sonella ozzardi microfilaremia (26%) was found in 1997,
whereas in 1999, a 3% point prevalence for anti–Trichinella spiralis antibodies, and a high seroreactivity against Taenia solium and Toxocara canis antigens were detected in the population living in this area.16,17
Meanwhile, no specific interventions were implemented against these infections.
The capacity of helminth infection to modulate host im-mune response, and thus their impact on allergic and/or
inflammatory diseases, has been a matter of a large volume
of research since the 1970s.18 The “hygiene hypothesis”
links the reduction in endemic infections due to housing, sanitation, and health improvements in high-income coun-tries with the concomitant rise in the prevalence of asthma and atopy in childhood.19In brief, the removal of the regu-latory effect of microorganisms and other parasites, after millennia of coevolution and genetic adaptation with hu-mans and other mammalian, might produce an imbalance in
the host pro- and anti-inflammatory immune responses and
an excess of immune-mediated diseases. However, little consensus exists among the studies that have explored the interaction between helminth infection and allergic
dis-eases, including meta-analysis and systematic reviews.20
For instance, as for the development of asthma, hookworm infection showed a strong protective role, but T. trichiura, Enterobius vermicularis, and S. stercoralis did not have an impact, whereas A. lumbricoides infection resulted a sig-nificant risk factor.21 Most of the published studies agree
that helminth infection protects against atopic sensitization and allergic skin reactivity, although these infections ap-pear associated with increased allergen-specific IgE.18,22 In recent years, deliberate infection of patients with worms has been proposed in non-endemic, high-income countries,
as a possible treatment for inflammatory diseases, such
as allergies, inflammatory bowel diseases, multiple sclero-sis, rheumatoid arthritis, and psoriasclero-sis, with inconsistent results.23
TABLE2
Serum concentrations of IgE (kU/L) and sCD30 (U/mL) of the human population surveyed in 1987 and 2013 in the Bolivian Chaco
Javillo (1987) Ivamirapinta (2013) P-value
Serum IgE, mean (SD) Total 205.4 (± 55.8) 77.9 (± 21.3) P < 0.001 SAC 206.3 (± 54.7) 79.9 (± 21.0) P < 0.001 < 26 years 199.1 (± 49.6) 80.0 (± 22.0) P < 0.001 ³ 26 years 220.7 (± 67.6) 76.3 (± 20.8) P < 0.001 Serum IgE, median (IQR) Total 186.0 (173.0–223.0) 81.5 (64.2–92.7) P < 0.001 SAC 188.0 (176.0–225.0) 84.0 (71.0–92.0) P < 0.001 < 26 years 186.0 (172.2–222.5) 84.0 (65.0–93.0) P < 0.001 ³ 26 years 186.0 (178.5–275.0) 76.0 (62.0–92.0) P < 0.001 Serum sCD30, mean (SD) Total 46.0 (± 19.2) 18.4 (± 10.6) P < 0.001 SAC 49.8 (± 19.7) 15.5 (± 7.2) P < 0.001 < 26 years 47.4 (± 18.4) 16.6 (± 7.6) P < 0.001 ³ 26 years 42.7 (±21.3) 19.7 (± 12.3) P < 0.001 Serum sCD30, median (IQR) Total 41.0 (33.0–57.0) 15.0 (11.0–25.0) P < 0.001 SAC 48.0 (34.0–61.0) 13.0 (11.0–17.0) P < 0.001 < 26 years 43.0 (36.0–57.7) 14.0 (11.0–24.0) P < 0.001 ³ 26 years 36.0 (27.5–52.5) 16.0 (11.0–27.0) P < 0.001
IQR = interquartile range; SAC = school-age children (4–15 years old); sCD30 = soluble CD30.
Both overall and age-stratified comparison showed a significant decline from 1987 to 2013 (P < 0.001) (Student’s t-test). No significant differences were found among the different age classes either in the 1987 or 2013 cohort.
FIGURE1. Box-plot of serum median IgE (left panel) and sCD30 (right panel) concentration distributions in patients from Javillo (1987) and
This study is not without limitations. One is that the two
surveys took place in different communities—however, the
Bolivian Chaco is a homogeneous ecological zone, where rural populations share the same hygienic and sanitary con-ditions, and no substantial differences in infestation rates are expected between two neighboring communities. Another limitation is that no parasitological data are available from Javillo—however, a parasitological survey conducted in 1987 in different rural communities of the Chaco region showed a
homogenously high STH prevalence.5A third concern would
be that the population of the second survey is old-er—however, different analyses concur to show the observed changes are age independent. Last and importantly, no information on allergic symptoms experienced by the partic-ipants and other epidemiological information on allergy-related clinical syndromes or immune-mediated diseases in the population are available. Further studies are warranted to address the clinical and epidemiological correlation of the serological rearrangements in populations where STH in-festation has virtually disappeared. Longitudinal studies car-ried out in settings where mass deworming programs are ongoing may help elucidate whether the removal of helminth infections would lead to an increased prevalence of allergic and other immune-mediated diseases in the long term.
Received March 4, 2019. Accepted for publication November 16, 2019.
Note: Supplemental table appears at www.ajtmh.org.
Funding statement: This work was supported by the University of Florence and the Italian Ministry of University and Research. Authors’ addresses: Chiara Della Bella, Michele Spinicci, Alessia Grassi, Marisa Benagiano, Simona Tapinassi, Heba Alnwaisri, Annalisa Azzurri, Mario Milco D’Elios, and Alessandro Bartoloni, Dipartimento di Medicina Sperimentale e Clinica, Universit `a degli Studi di Firenze, Firenze, Italy, E-mails: chiara.dellabella@unifi.it, michele.spinicci@unifi.it, alessia.grassi@unifi.it, marisa.benagiano@ unifi.it, simona.tapinassi@unifi.it, hebafmustafa.alnwaisri@unifi.it, aazzurri@usl4.toscana.it, mariomilco.delios@unifi.it, and alessandro. bartoloni@unifi.it. David Rojo, Escuela de Salud del Chaco Tekove Katu, Gutierrez, Plurinational State of Bolivia, E-mail: davidrojomayaregua@gmail.com. Herlan Gamboa, Facultad Integral del Chaco, Universidad Aut ´onoma Gabriel Ren ´e Moreno, Camiri, Plurinational State of Bolivia, E-mail: herlan.gamboa@hotmail.com. Roberto Torrez and Joaqu´ın Monasterio, Servicio Departamental de Salud (SEDES) de Santa Cruz, Santa Cruz, Plurinational State of Bolivia, E-mails: dr_torresfernandez@hotmail.com and joaquinmpinckert@ hotmail.com. Simona Gabrielli and Gabriella Cancrini, Dipartimento di Sanit `a Pubblica e Malattie Infettive, Universit `a di Roma Sapienza, Roma, Italy, E-mails: simona.gabrielli@uniroma1.it and gabriella. cancrini@uniroma1.it. Fabio Macchioni, Dipartimento di Scienze Veterinarie, Universit `a degli Studi di Pisa, Pisa, Italy, E-mail: fabio. macchioni@unipi.it. Antonio Montresor, Department of Control of Neglected Tropical Diseases (NTD), World Health Organization, Ge-neva, Switzerland, E-mail: montresora@who.int. Piero Olliaro, Nuffield Department of Medicine, Centre for Tropical Medicine and Global Health, University of Oxford, Oxford, United Kingdom, E-mail: piero. olliaro@ndm.ox.ac.uk.
REFERENCES
1. WHO, 2017. Integrating Neglected Tropical Diseases into Global Health and Development: Fourth WHO Report on Neglected Tropical Diseases. Geneva, Switzerland: World Health Organi-zation, Available at: https://www.who.int/neglected_diseases/ resources/9789241565448/en/. Accessed February 12, 2019. 2. Bisoffi Z et al., 2013. Strongyloides stercoralis: a plea for action.
PLoS Negl Trop Dis 7: e2214.
3. World Health Organization, 2001. Schistosomiasis and Soil-Transmitted Helminth Infections. Fifty-Fourth World Health As-sembly. Ninth Plenary Meeting. WHA54.19. Available at: http:// apps.who.int/gb/archive/e/e_wha54.html. Accessed February 12, 2019.
4. WHO, Schistosomiasis and Soil-Transmitted Helminthiases: Num-ber of People Treated in 2017. Weekly Epidemiological Record 50; 2018, 93 681–692. Available at: https://www.who.int/ neglected_diseases/resources/who_wer9350/en/. Accessed February 13, 2019.
5. Cancrini G, Bartoloni A, Paradisi F, Nunez LE, 1989. Parasito-logical observations on three Bolivian localities including rural communities, cities and institutions. Ann Trop Med Parasitol 83: 591–594.
6. Bartoloni A, Guglielmetti P, Cancrini G, Gamboa H, Roselli M, Nicoletti A, Paradisi F, 1993. Comparative efficacy of a single 400 mg dose of albendazole or mebendazole in the treatment of nematode infections in children. Trop Geogr Med 45: 114–116. 7. Macchioni F, Segundo H, Gabrielli S, Totino V, Gonzales PR, Salazar E, Bozo R, Bartoloni A, Cancrini G, 2015. Dramatic decrease in prevalence of soil-transmitted helminths and new insights into intestinal protozoa in children living in the Chaco region, Bolivia. Am J Trop Med Hyg 92: 794–796.
8. Spinicci M et al., 2018. Scaling down of a deworming programme among school-age children after a thirty-year successful in-tervention in the Bolivian Chaco. Trop Med Int Health 23: 616–621.
9. Spinicci M et al., 2017. Seroepidemiological trend of strongyloi-diasis in the Bolivian Chaco (1987–2013) in the absence of disease-specific control measures. Trop Med Int Health 22: 1457–1462.
10. D’Elios MM, Benagiano M, Della Bella C, Amedei A, 2011. T-cell response to bacterial agents. J Infect Dev Ctries 5: 640–645. 11. D’Elios MM et al., 1997. In vivo CD30 expression in human
dis-eases with predominant activation of Th2-like T cells. J Leukoc Biol 61: 539–544.
12. Smits HH, Everts B, Hartgers FC, Yazdanbakhsh M, 2010. Chronic helminth infections protect against allergic diseases by active regulatory processes. Curr Allergy Asthma Rep 10: 3–12. 13. Alexandre-Silva GM, Brito-Souza PA, Oliveira ACS, Cerni FA, Zottich U, Pucca MB, 2018. The hygiene hypothesis at a glance: early exposures, immune mechanism and novel therapies. Acta Trop 188: 16–26.
14. Lienhardt C et al., 2002. Active tuberculosis in Africa is associated with reduced Th1 and increased Th2 activity in vivo. Eur J Immunol 32: 1605–1613.
15. Macchioni F, Segundo H, Totino V, Gabrielli S, Rojas P, Roselli M, Paredes GA, Masana M, Bartoloni A, Cancrini G, 2016. In-testinal parasitic infections and associated epidemiological drivers in two rural communities of the Bolivian Chaco. J Infect Dev Ctries 10: 1012–1019.
16. Bartoloni A, Cancrini G, Bartalesi F, Marcolin D, Roselli M, Arce CC, Hall AJ, 1999. Mansonella ozzardi infection in Bolivia: prevalence and clinical associations in the Chaco region. Am J Trop Med Hyg 61: 830–833.
17. Nicoletti A et al., 2002. Epilepsy, cysticercosis, and toxocariasis: a population-based case-control study in rural Bolivia. Neurol-ogy 58: 1256–1261.
18. Santiago HC, Nutman TB, 2016. Human helminths and allergic disease: the hygiene hypothesis and beyond. Am J Trop Med Hyg 95: 746–753.
19. Bach JF, 2002. The effect of infections on susceptibility to auto-immune and allergic diseases. N Engl J Med 347: 911–920. 20. McSorley HJ, Chay ´e MAM, Smits HH, 2019. Worms: pernicious
parasites or allies against allergies? Parasite Immunol 41: e12574. 21. Leonardi-Bee J, Pritchard D, Britton J, 2006. Asthma and current intestinal parasite infection: systematic review and meta-analysis. Am J Respir Crit Care Med 174: 514–523.
22. Feary J, Britton J, Leonardi-Bee J, 2011. Atopy and current in-testinal parasite infection: a systematic review and meta-analysis. Allergy 66: 569–578.
23. Wammes LJ, Mpairwe H, Elliott AM, Yazdanbakhsh M, 2014. Helminth therapy or elimination: epidemiological, immuno-logical, and clinical considerations. Lancet Infect Dis 14: 1150–1162.
